Influence of testosterone on regulation of ODC, antizyme, and N 1 -SSAT gene exp - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:44 |只看该作者 |倒序浏览 |打印

作者：Olivier Levillain, Anna Greco, Jean-Jacques Diaz, Roger Augier, Anne Didier, Karine Kindbeiter, Frédéric Catez, and Myriam Cayre作者单位：1 Laboratoire de PhysiopathologieMétabolique et Rénale, Faculté de Médecine Lyon R.T. H. Laënnec, Institut National de la Santé et de la RechercheMédicale, Unite 49 69372 Lyon Cedex 08; 2 Centre de Génétique Moléculaireet Cellulaire ) U( [% F8 ~7 n( ]3 s





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      【摘要】
   Polyamines are involved in the control of the cell cycle and cell growth.In murine kidney, testosterone enhances gene expression of ornithinedecarboxylase (ODC), the first enzyme in polyamine biosynthesis. In thisstudy, we document the time course effect of testosterone on 1 ) geneexpression of ODC, antizyme 1 (AZ1), andspermidine/spermine- N 1 -acetyltransferase( N 1 -SSAT); 2 ) ODC activity in proximal convolutedtubules (PCT) and cortical proximal straight tubules (CPST); and 3 )renal polyamine levels. Female mice were treated with testosterone for aperiod of 1, 2, 3, and 5 consecutive days. ODC gene expression was extremely low in kidneys of untreated female mice compared with that of males.Consequently, the renal putrescine level was sevenfold lower in females thanin males, whereas spermidine and spermine levels did not differ between sexes.In female kidneys, testosterone treatment sharply increased ODC mRNA andprotein levels as well as ODC activity. Testosterone increased the expression of ODC in PCT and CPST over different time courses, which suggests that ODCactivity is differentially regulated in distinct tubules. The expression ofAZ1 and N 1 -SSAT mRNA was similar in male and female mousekidneys. Testosterone treatment enhanced AZ1 and N 1 -SSATmRNA levels in a time-dependent manner by unknown molecular mechanisms.Putrescine and spermidine levels increased after testosterone treatment infemale kidneys. Surprisingly, although ODC protein and activity wereundetectable in female kidneys, the levels of AZ1 mRNA and protein weresimilar to those in males. Therefore, one may propose that ODC protein couldbe continuously degraded by AZ1 in female kidneys. % [3 l" B( C) ~6 t
      【关键词】 isolated tubules proximal tubule permeabilized tubules gene expression regulation6 L/ `$ A: {* K) ~- `& |
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THE NATURAL ALIPHATIC POLYAMINES, putrescine (Put), spermidine (Spd), and spermine (Spm), are ubiquitously found in most animal and planttissues ( 30 ). Polyamines areinvolved in many cellular and physiological processes including cell growthand differentiation ( 10 ) andother biological functions that have not yet been fully elucidated. Forexample, these polycations interact with a great variety of negatively chargedentities inside cells, such as nucleic acids, membranes, ribosomes, lipids,and other small molecules.
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Biosynthesis of Put, Spd, and Spm requires several enzymes: ornithinedecarboxylase (ODC; EC 4.1.1.17  ), L -methionine adenosyltransferase (EC 2.4.2.13  ), S -adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50  ), spermidine synthase (EC 2.5.1.16  ), and spermine synthase (EC2.5.1.22  ) ( 30, 38 ). In the interconversionpolyamine pathway, Spm is successively converted into Spd and Put. Polyamine catabolism is controlled by two enzymes,spermidine/spermine- N 1 -acetyltransferase ( N 1 -SSAT) and FAD-dependent polyamine oxydase (PAO; EC1.5.3.11  ) ( 38 ). Becausepolyamine accumulation is cytotoxic, the polyamine pathway has to be highlyregulated to finely control the intracellular concentration of polyamines.Excesses of acetylated and nonacetylated polyamines are excreted in urine.$ f" h! P( ?" h' v

Enzymes involved in polyamine metabolism, such as ODC, SAMDC, and PAO, areknown to be especially abundant in the male mouse kidney( 7, 12, 14, 22 ). ODC mRNA, protein, andenzyme activity have been clearly localized by various techniques in the renal cortex, particularly in the proximal convoluted tubule (PCT) of the male mouse( 7, 18, 21, 31 ). In contrast, all are extremely low in the female mouse kidney. Recently, in a detailed study, noODC activity was found in isolated nephron segments dissected from femaleSwiss mice ( 20 ). Therefore,the female mouse kidney constitutes an excellent tool for studying in detail factors involved in the regulation of the polyamine pathway by sexhormones.

Among the numerous factors regulating polyamine biosynthesis ( 9, 34, 35 ), androgens such astestosterone dramatically increase ODC mRNA, protein levels, and ODC activityand cause Put to accumulate in kidneys of male and treated female mice ( 1, 7, 12, 18, 20, 29 ). Conversely, in castratedmales, ODC gene expression decreases and reaches the levels observed infemales ( 29 ). However, atpresent, the influence of testosterone on the regulation of the entirepolyamine pathway remains unknown.

The polyamine pathway is highly regulated at transcriptional, translational, and posttranslational levels. Regulation of ODC expression hasbeen well documented, including the original feedback controlled by theprotein antizyme 1 (AZ1) ( 15, 27 ). AZ1 has a high affinityfor ODC and binds reversibly to the proline-, glutamic acid-, serine-, andthreonine-rich (PEST) region of ODC( 11, 26 ). The PEST regioncorresponds to a signal for selective proteolysis and is presumed to beinvolved in the degradation of proteins having rapid turnover. When the ODC-AZcomplex binds to the 26S proteasome for degradation, AZ1 molecules arereleased and recycled. AZ1 plays a central role in the negative-feedbacksystem by accelerating ODC degradation and preventing polyamineoveraccumulation. In addition, AZ1 negatively regulates the cellular polyaminelevels by inhibiting a specific transporter involved in polyamine uptake( 28 ).1 |6 e( Y- V4 q! E8 e/ o' s/ x

The present study was designed 1 ) to analyze in detail the time course effect of testosterone on ODC gene expression and ODC activity insingle microdissected proximal tubules, 2 ) to resolve whether renalAZ1 and N 1 -SSAT gene expression differs between male andfemale mice, 3 ) to study the time course influence of testosterone onAZ1 and N 1 -SSAT gene expression, and 4 ) toquantify polyamines in kidneys of control mice and testosterone-treated femalemice.

MATERIALS AND METHODS
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Animals and Treatments

Nine-week-old adult female [30-32 g body wt (BW)] and male OF-1 Swiss (IOPSCaw) mice (35-40 g BW) from Iffa Credo (L'Arbresle sur Orge, France) had freeaccess to tap water and a standard laboratory diet (Souffirat 20% protein,Genthon). The institutional animal care committee approved allexperiments.; C" e4 K8 v4 R
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Female mice were subdivided into six groups: one untreated group (control),one oil-treated group, and four androgen-treated groups. Mice subjected totestosterone treatment were injected subcutaneously with 150 µltestosterone propionate (31 mg/ml in sesame oil, i.e., 155 µg/g BW).Injections were performed at 8:00 A.M., and mice were treated for a period of 1, 2, 3, or 5 consecutive days. Oil-treated mice were injected subcutaneouslywith 150 µl sesame oil for a period of 5 consecutive days. Untreated malemice were used as positive controls.
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Sampling of Kidneys for Northern and Western Blot Analyses

Twenty-four hours after the last injection of testosterone or oil, micewere anesthetized (ip) with 0.1 ml/100 g BW pentobarbital sodium (Nembutal,6%; Clin Midy, Paris, France) diluted 1:2 in 0.9% NaCl solution. Control micewere similarly anesthetized. The left and right kidneys were rapidly removedand decapsulated. The blood contained in each kidney was immediately removed with blotting paper. The kidneys were placed in a sterilized Eppendorf tubeand frozen in liquid nitrogen. They were maintained at -80°C until RNA orprotein extractions.

RNA Extraction and Northen Blot Analyses of ODC, AZ1,N 1 -SSAT, and -Actin mRNA$ f! l5 l9 V) m3 l
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Each frozen kidney was homogenized in 3 ml RNAxel solution (Eurobio), andtotal RNAs were extracted according to the manufacturer's recommendations andmaintained at 4°C. RNAs were rinsed twice with 70% ethanol and dried in aSpeed Vac. RNAs were resuspended in cold 10 mM Tris·HCl and 1 mM EDTA,pH 8.0, and their concentrations were determined by absorbance at 260 nm.Fifteen micrograms of RNA samples were submitted to 1.2% agarose gelelectrophoresis. After the gel was treated for 20 min in 50 mM NaOH, then for20 min in a solution containing 0.5 M Tris and 1.5 M NaCl, RNAs weretransferred overnight to a nylon membrane (Appligene) and immobilized using aUV cross-linker (Appligene).
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Membranes were hybridized with murine 32 P-labeled cDNAscorresponding to ODC [pBS-ODC Xho I- Bam HI( 20 )], AZ1 [pcDNA3.1 ( ) WTAZ, Eco RI- Eco RI( 15 )], and -actin[pAL41-cytoplasmic -actin, Pst I- Pst I, ( 8 )] and human 32 P-labeled N 1 -SSAT cDNAs [pBluescript SAT9.3, Eco RI- Eco RI( 24 )]. cDNA were labeled usingthe RTS RadPrime DNA labeling system (GIBCO BRL, Life Technologies) and -[ 32 P]dCTP. Hybridization was performed overnight at65°C. Membranes were washed three times in 2 x SSC (0.3 M NaCl and 30mM sodium citrate), 5 mM phosphate buffer, and 0.1% SDS and three times in0.5 x SSC, 3 mM phosphate buffer, and 0.1% SDS. The amount ofradioactivity hybridized to specific mRNA was estimated after scanningdensitometry of the membranes using a PhosphorImager SI (Molecular Dynamics,Amersham). Quantitation of -actin mRNA was used as a control of equalloading and RNA transfer.5 C5 O7 E' x6 o: s' V
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Protein Extraction and Western Blot Analyses of ODC and AZ1Proteins
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Each frozen kidney was mixed at 4°C with a Turrax in 1 ml of lysingbuffer ( 19 ) containing 1 mMprotease inhibitor cocktail, 1 mM benzamidine, and 1 mM PMSF and thencentrifuged at 10,000 g for 30 min at 4°C. Protein concentrationswere determined in the supernatant using the Bradford protein assay( 5 ). Fifty-microgram samples ofsoluble proteins were subjected to 12% PAGE containing SDS using 6 W/gel andtransferred to a polyvinylidene difluoride membrane (0.45 µm, Immobilon-P,Millipore) at 150 mA for 90 min. Proteins were fixed on the membrane withPonceau S solution for 15 min. Immunoblots were washed twice in 1 x Tris-buffered salt 0.15% Tween 20 (TBST) and immersed in a blocking solutionconsisting of 5% fat-free milk powder in 1 x TBST for 30 min.2 O7 h  D2 U) y4 n

The blots were incubated with a polyclonal rabbit anti-human ODC(Eurodiagnostica), polyclonal rabbit anti-rat AZ1 [a gift of Dr. S. Matsufuji,Tokyo, Japan ( 23 )], ormonoclonal mouse anti- -tubulin primary antibodies in 5% milk-1 x TBST. Blots were washed three times for 10 min in 1 x TBST and incubated for 60 min with either peroxidase-conjugated anti-rabbit IgG or anti-mouse IgGsecondary antibodies or an anti-rabbit IgG secondary antibody conjugated toalkaline phosphatase in 5% milk-1 x TBST. Blots were washed three timesfor 10 min in 1 x TBST, and antibody binding was revealed using either anenhanced chemifluorescence (ECF) or ECL Western Blotting Kit. ECL detection was performed using Kodak X-MAT film. Low-exposure film was scanned, and theintensity of the bands was estimated using the ImagerMaster Total Lab v1.00program (Pharmacia, Orsay, France). The intensity of the bands detected by ECFwas estimated after scanning densitometry of the membranes with a FluoroImager SI (Molecular Dynamics, Amersham).  y) W7 O5 P7 ?
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Kidney Preparation and Microdissection of Different NephronSegments
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Female mice were anesthetized as described above, and the left kidney wasprepared for microdissection as previously described ( 20, 21 ). The kidney was perfusedwith 4 ml of medium for incubation and perfusion (MIP; see below) containing1.63 mg/ml collagenase, removed, and sliced along the corticomedullary axis.Small pyramids containing both cortical and medullary tissue were incubated at30°C in MIP containing 0.9 mg/ml collagenase, bubbled with O 2,and shaken at 60 cycles/min for a period of 5-10 min.

MIP consisted of (in mM) 137 NaCl, 5 KCl, 0.44KH 2 PO 4, 1 MgCl 2, 0.8 MgSO 4, 0.33Na 2 HPO 4, 1 CaCl 2, and 20 HEPES, as well as 0.1% BSA, 1% vitamin mixture, 6% dextran, and energy-providing substrates [(inmM) 5 glucose, 5 lactate, 10 acetate, 1 pyruvate, and 2 glutamine] and wasadjusted to pH 7.4 with NaOH. The osmolarity was 350 mosmol/kgH 2 O.Before use, MIP was bubbled with O 2.
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Pyramids of tissues were rinsed in MIP. PCT, cortical proximal straighttubule (CPST), and cortical thick ascending limb (CTAL) were microdissected at4°C in MIP using a stereomicroscope. Tubules were transferred ontosiliconized, BSA-coated hollow glass slides with 0.5 µl MIP and tightlysealed with a glass coverslip. The tubules were drawn under a microscope witha clear chamber for subsequent length measurement. Samples were kept at4°C until Triton X-100 treatment and metabolic incubation.4 ?  U' C. @& i% c; b, j

Measurement of ODC Activity in Permeabilized Nephron Segments
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ODC activity was quantitated according to a previously validated technique( 20, 21 ). PCT, CPST, and CTAL wereobtained from 10 control and 21 testosterone-treated mice as described above. Tubules were permeabilized by adding a 0.5-µl droplet of the following2 x buffer: 0.05% Triton X-100 (wt/vol) and (in mM) 40 HEPES, 60NaH 2 PO 4, 0.2 pyridoxal-5-phosphate (PLP), 0.2 EDTA, and10 DTT (205 mosmol/kgH 2 O, pH adjusted to 7.40). TritonX-100-treated tubules were maintained for 60 min at 4°C. CTAL were used toverify the efficiency of the Triton X-100 treatment, which abolishes ornithineoxidation. Incubation was started by the addition of 1 µl of the samebuffer (1 x ), which contained 100 µM L -[1- 14 C]ornithine (460 Bq/sample; 1.85 MBq/µmol; pH7.40). The incubating chamber was sealed with a glass coverslip containing a2-µl droplet of KOH. Samples were incubated in a water bath at 37°C for 70 min. The KOH droplet containing the 14 CO 2 wasremoved, and the amount of radioactivity was estimated by liquid scintillation counting. Control or blank samples contained labeled L -[1- 14 C]ornithine, but no tubules. Results areexpressed in femtomoles of 14 CO 2 produced per minute permillimeter tubular length.
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Polyamine Extraction and Quantitation of Put, Spd, and Spm

The whole right blood-free kidney was weighed, and polyamines wereextracted by homogenizing each frozen tissue in 1 ml of ice-cold 0.4 NHClO 4 /100 mg tissue. The samples were centrifuged at 4,000 g for 30 min at 4°C, and the clear supernatant was collected andstored at -20°C until further analysis.2 u, e% F" S6 |5 k7 z  A; `- @* i& v% Q

Tissue extracts and standards [Put, Spd, Spm, and diaminoheptane (DAH) asan internal standard] were dansylated according to the procedure described bySeiler ( 37 ) and were thentreated using the protocol adapted from Besson et al.( 2 ). Dansylation was assessedin glass vials by mixing 40 µl of HClO 4 extracts, 10 µl ofDAH, and 100 µl of 0.3 M Na 2 CO 3 (Merck). The reactionwas initiated by adding 200 µl of freshly prepared dansyl chloride solution(5 mg/ml) in acetone (SDS, spectrosol grade) and allowed to proceed overnightin the dark at room temperature. After dansylation, each sample was dilutedwith 700 µl H 2 O, vortexed, and applied to a Waters Sep-PakC 18 cartridge ( 4 ).After being washed with 4 ml of 20% methanol, the polyamine-containingfraction was eluted with 2 ml of 100% methanol. The separation andquantification of polyamines were performed by reverse-phase (RP)-HPLC using aWaters system composed of two model 510 pumps, a Wisp 700 autosampler, and anNEC APC4 data module recorder integrator. A Merck F 1050 fluorescencespectrophotometer was used to detect fluorescence (350-nm excitation and495-nm emission). The separations were performed on an RP18 Merck Lichrocart(25 x 4 mm, 5 µm) precolumn and an RP18, 100 CH Merck column (125 x 4 mm, spherical packing, 5 µm). The solvent system was anacetonitrile/water gradient at a flow rate of 1 ml/min of 60% acetonitrile in water for 7 min and then 90% acetonitrile for 10 min and a 98% acetonitrilepurge for 5 min. The column was reequilibrated to the initial 60% acetonitrileconditions for a 10-min period between successive injections. For eachdetermination, 50 or 100 µl of dansylated samples were injected into theequilibrated column.) z- Y' e, `# H: u

The major polyamines were identified by their retention times compared withthose of standard (10-70 pmol) polyamines. Peak areas were automaticallymeasured by the integrator and evaluated according to the calibration method( 4 ). The absolute limit ofdetection per injection was 1 pmol of dansylated Spd and dansylated Spm and 7pmol of dansylated Put, respectively. Two blank injections were routinely runbetween calibrations and sample analysis.* p" s% a# V- m& P; v6 k

Chemicals

Salts, Ponceau S solution, secondary antibodies, Kodak X-MAT film, DAH, anddansyl chloride were purchased from Sigma (St. Quentin Fallavier, France).Collagenase A from Clostridium histolyticum (0.28 U/mg) and proteaseinhibitor cocktail were from Boehringer Mannheim (Strasbourg, France). L -[1- 14 C]ornithine (1.85 GBq/mmol) and -[ 32 P]dCTP (220 TBq/mmol), monoclonal mouse anti- -tubulin, and ECF and ECL Western Blotting Kits were purchased fromAmersham (Buckinghamshire, UK, and Orsay, France).5 ~9 N& y' W* i8 \# S6 R! v

Statistical Analyses

Results are presented as means ± SE. Statistically significant differences were calculated either by an unpaired Student's t -test orby one-way ANOVA and the Tukey-Kramer test (StatView 5) at the 95% levelsignificance where appropriate.

RESULTS
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The purpose of these experiments was to study the influence of testosteroneon ODC, AZ1, and N 1 -SSAT gene expression in the murinekidney. To achieve this goal, female mice were injected with testosteronepropionate for a period of 1, 2, 3, or 5 consecutive days. Groups of untreatedmale and female mice as well as female mice treated with oil for 5 days wereused as controls. RNAs, proteins, and polyamines were extracted from the wholekidney and analyzed, whereas ODC activity was quantified in single, isolatedproximal tubules.. h$ Q( D) \  }) M! {) {  W
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Effect of Testosterone on ODC Gene Expression and Activity0 j, C$ z. ~2 W& {2 X4 _

Time course effect of testosterone on ODC mRNAs in female mouse kidney. ODC mRNA levels were determined to assess the ability oftestosterone to regulate ODC gene expression and to establish the time coursevariation of their induction. Using a specific ODC cDNA probe, two forms ofODC mRNAs (2.2 and 2.7 kb) were detected by Northern blotting in the kidneysof untreated male and female mice and testosterone-treated female mice( Fig. 1 ) ( 16 ). By contrast, the 2.2-and 2.7-kb ODC mRNAs were barely detectable in untreated female kidneyscompared with male kidneys. After a single injection of testosterone intofemale mice, the level of the two renal ODC mRNAs reached that in males. Prolonged testosterone treatment led to a progressive increase in 2.2- and2.7-kb ODC mRNA levels before a plateau was reached after 2-3 days.Testosterone treatment had no effect on the content of control -actinmRNA in the kidney ( Fig.1 ).
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Fig. 1. A : Northern blot analysis of ornithine decarboxylase (ODC) and -actin mRNA levels in male and untreated and testosterone-treated femalemouse kidneys. Female mice were injected subcutaneously with 150 µltestosterone propionate (31 mg/ml in sesame oil). Injections were performedevery day at 8:00 A.M. for a period of 1, 2, 3, or 5 consecutive days. Eachline corresponds to 15 µg total RNA extracted from one mouse kidney. B : quantitation of ODC and -actin mRNA levels. The amount ofradioactivity hybridized was estimated after scanning densitometry of themembranes using a PhosphorImager. Left : 2.2 kb- and 2.7-kb ODC mRNAs. Right : 2.1-kb -actin mRNA. Untreated male (M) and female (F; day 0 ) mice were used as controls for ODC mRNA. -Actin mRNA wasused as a control. Values are means ± SE, except when n =2.1 v0 r/ n4 K4 w' [8 N
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Time course effect of testosterone on ODC protein level in female mousekidney. Western blotting was performed to monitor the amount of ODCprotein. An 53-kDa protein, which corresponds to the predicted size of theODC polypeptide subunit (Swiss-Prot: mouse P00860  ), was specifically revealedin kidneys of male and treated female mice using a specific anti-ODC antibody ( Fig. 2, left ). -Tubulin, a 55-kDa protein, was used as a control of loading andtransfer of proteins ( Fig. 2, right ). Immunoblots were revealed by ECF to estimate ODC proteinlevels during the course of treatment. No ODC protein was detectable inkidneys of control untreated female mice. ODC protein levels increased progressively after testosterone injection and reached a peak between days3 and 5 ( Fig. 2 ).The levels of ODC mRNAs are therefore tightly correlated with those of ODCprotein in kidneys of male, female, and testosterone-treated female mice(Figs. 1 and 2 ).; W' u( |7 l( Y/ R

Fig. 2. Western blot analysis of ODC and -tubulin protein levels in kidney ofmale and untreated and testosterone-treated female mice. Mice were treated asindicated in the legend to Fig.1. Each lane corresponds to 1 mouse and 50 µg of solubleprotein. Left : representative immunoblot probed with a rabbit antihuman-ODC antibody and revealed by chemiluminescence (ECL) after exposure toX-ray film. The antibodies revealed a protein at 53 kDa, which corresponds toODC, and revealed a single band of 55 kDa, which corresponds to -actin,as expected. Right : ODC protein levels, as revealed by fluorescence(ECF) and quantification using a FluoroImager SI and a computer-assistedimager-analyzer. -Tubulin was used as a control of protein loading andtransfer. Values are means ± SE; n = 3 for male and untreatedfemale mice. For the others, n = 2.3 [# e; C2 }  {0 u7 J

Time course effect of testosterone on ODC activity in proximal tubules. Numerous investigators have reported an increase in renal ODCactivity in mice after testosterone injection( 12, 25, 33 ). However, it has neverbeen determined whether the time course of the testosterone-induced ODCactivity differed from one tubule to another. We measured ODC activity insingle microdissected PCT and CPST isolated from testosterone-treated femalemice ( 20 ). In untreated femalemice ( day 0 ), ODC activity was undetectable in any tubule( Fig. 3 ). In contrast, intestosterone-treated females, ODC dramatically increased in both PCT and CPST.However, the time course of ODC activity differed between PCT and CPST. InPCT, ODC activity increased quite linearly during the course of the hormonaltreatment ( Fig. 3 ), whereas inCPST it increased sharply from day 1 to day 3 and thenremained constant until day 5 ( Fig. 3 ). Although the highestbasal level of ODC activity was found in the PCT of male mice( 21 ), in females treated withtestosterone for 2, 3, and 5 days, ODC activity was 38% higher in CPST than inPCT (unpaired Student's t -test, P & U. f0 r9 [6 D; a! ~
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Fig. 3. Time course effect of testosterone on ODC activity in single microdissectedproximal convoluted (PCT) and cortical proximal straight (CPST) tubules. Micewere treated as indicated in the legend to Fig. 1. Triton X-100-treatedtubules were incubated with 100 µM L -[1- 14 C]ornithine. Values are means ± SE, withthe no. of samples tested shown in parentheses; n = 10 and 13 mice incontrol and day 5, respectively, and n = 3-5 mice in theother groups. * Difference in ODC activity between PCT and CPST fora given day ( P 1 C5 I5 P- z, ]% _- v& E
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Effects of Testosterone on Polyamine Content of the Kidney5 L( p7 D1 w( }6 [
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Quantitation of Put, Spd, and Spm in untreated mouse kidneys revealed sexdifferences. Put content was sevenfold higher in male than in female kidneys( Fig. 4, 1-way ANOVA, F = 33.8, P / Z# u9 H" F" K& K
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Fig. 4. Putrescine (Put), spermidine (Spd), and spermine (Spm) contents in thekidneys of male and untreated, oil-treated, and testosterone-treated femalemice. Mice were treated as indicated in the legend to Fig. 1. Values are means± SE and were analyzed for statistical significance by 1-way ANOVA andthe Tukey-Kramer test; ( n ) = males( 6 ); untreated( 6 ) and oil-treated( 4 ) females; and femalestestosterone treated for 1 day( 6 ) or 2( 7 ), 3( 6 ), and 5 days( 6 ). * P

Influence of Testosterone on AZ1 gene expression9 F+ F5 N% g- g

Analysis of the level of AZ1 mRNAs in kidneys of control untreated andtestosterone-treated mice. As testosterone dramatically enhanced ODC geneexpression and shifted renal polyamine concentrations to high levels, theinfluence of testosterone on AZ1 mRNA and protein content was investigated.Indeed, polyamines are involved in AZ mRNA translation by increasing thefrequency of the frame shift, which enables the synthesis of the whole AZprotein ( 27 ).

Northern blot analyses using a specific AZ1 probe revealed the presence ofa single 1.3-kb AZ1 mRNA in the kidneys of untreated male, untreated female,and testosterone-treated female mice ( Fig.5 A ). Similar levels of AZ1 mRNA were detected inuntreated male and female kidneys. However, in kidneys of testosterone-treated female mice, AZ1 mRNA levels progressively increased during the course of thetreatment. On day 5 of treatment, the amount of AZ1 mRNA was abouttwofold higher than that found in untreated female mice( Fig. 5 A ).% I2 D- I* i8 a0 W: `# p& k% k0 }
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Fig. 5. Determination of the levels of antizyme 1 (AZ1) mRNA ( A ) andproteins ( B ) in kidneys of male and untreated andtestosterone-treated female mice. Mice were treated as indicated in the legendto Fig. 1. A, left :AZ1 mRNAs detected by Northern blotting on the same membrane used to probe ODCand -actin mRNAs (see Fig.1 ). Right : quantitation of AZ1 mRNA levels, performed asindicated in the legend to Fig.1. Values are means ± SE for male ( n = 3),untreated ( n = 4), and testosterone-treated ( n = 3 for 5-daytreatment) female mice; n = 2 for the others. B, left :representative immunoblot probed with a rabbit anti-AZ1 antibody and revealedby ECL after exposure to X-ray film. The antibody revealed proteins at 24.1kDa (open and closed bars) and 26.5 kDa (hatched bars), which correspond toAZ1. Right : AZ1 protein levels, quantified as reported in Fig. 2. Each lane correspondsto 1 mouse and 100 µg soluble protein extracts. Samples are the same usedfor Fig. 2. Values are means± SE for males ( n = 3) and untreated and 5-day testosteronetreatment females ( n = 3); n = 2 for the others.: n3 ~9 U4 m) Q) s0 [! S8 S7 ?
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Analysis of the level of AZ1 protein in kidneys of control, untreated,and testosterone-treated mice. Expression of AZ1 protein in kidneys wasinvestigated by Western blot analysis. The specific anti-AZ1 antibody revealedproteins of different sizes ( Fig.5 B ), 24.1 kDa for the most abundant and 26.5 kDa for theleast abundant ( 27 ). Bothproteins exhibited the same pattern of expression and were abundant in bothuntreated male and female kidneys. Testosterone treatment slightly increased by 1.6-fold AZ1 protein levels in female mice.8 T# t( t: N  R6 ?2 `

Influence of Testosterone on N 1 -SSAT, a Key Enzyme in theRetroconversion Route% w! J) a, h' V3 N- G* B2 z9 `
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Analysis of N 1 -SSAT mRNAs in kidneys of control, untreated, and testosterone-treated female mice. The polyamine pathway is tightlyregulated to avoid the cytotoxic effect due to polyamine accumulation. As ODCgene expression was dramatically stimulated and polyamine levels were enhancedin testosterone-treated female mouse kidneys, one would expect theretroconversion route, controlled by the enzyme N 1 -SAT, tobe active. Northern blot analyses using a probe specific for N 1 -SSAT revealed a single 1.1-kb N 1 -SSAT mRNA in the kidney of all mice( Fig. 6 ). In untreated mice, N 1 -SSAT mRNA was barely detectable, contrasting with ourresults for ODC and AZ1 mRNAs. In female kidneys, testosterone progressivelyincreased N 1 -SSAT mRNA levels, which reached a plateau on day 3 of treatment ( Fig.6 ).9 X! K1 v  {4 j4 \: A. k
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Fig. 6. Analysis of spermidine/spermine- N 1 -acetyltransferase( N 1 -SSAT) mRNA levels in kidney of male and untreated andtestosterone-treated female mice as determined by Northern blotting. Mice weretreated as indicated in the legend to Fig.1. Left : N 1 -SSAT mRNAs were detectedon the same membrane than that used to probe ODC and -actin mRNAs (see Fig. 1 ). Right :quantitation of N 1 -SSAT mRNA levels, as estimated afterscanning densitometry of the membrane (as indicated in the legend to Fig. 2 ). Values are means± SE for male ( n = 3), untreated female ( n = 4), and5-day testosterone-treated female mice ( n = 3); n = 2 forthe others.
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DISCUSSION
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It has been known for several years that testosterone regulates the ODCgene in the mouse kidney. In some studies, ODC mRNA, protein, and enzymeactivity were analyzed in whole kidney extracts, thereby discarding the notionof the anatomic heterogeneity of the kidney( 13 ). In other reports, theuse of histological approaches, e.g., in situ hybridization,immunocytochemistry, and autoradiography, provided more insight into thelocalization of ODC in the kidney. Within the male nephron, the proximal tubule was identified as the only site containing ODC mRNA ( 7 ) and protein( 31, 36 ). Recent data have shownthat ODC activity is inequally distributed along the proximal tubule, as itdecreased sharply from the pars convoluta toward the terminal portion of thepars recta ( 21 ). By contrast,no ODC activity was detected along the female mouse nephron, even when a verysensitive radiolabeling method was used( 20 ). However, in female mice,androgens induced ODC gene expression, specifically in the whole cortex andthe outer stripe of the outer medulla, as visualized by in situ hybridization( 7, 20 ) and immunohistochemistry ( 17 ).6 z! @4 Z5 e! A* m& H! Z* J
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In this paper, we analyzed for the first time in single, isolated proximaltubules (a few hundred cells corresponding to 0.5- to 1-mm tubule)microdissected from kidneys of female mice, the time course of ODC activity,as induced by testosterone over 1-5 days. This fine approach revealed that theinduction of ODC activity differs temporally between the subsegments of theproximal tubule. In PCT, ODC activity increased quite linearly during thecourse of the hormonal treatment, whereas in CPST it increased sharply andprogressively during the first 3 days, then remained constant. Furthermore,ODC activity was higher in CPST than in PCT isolated from androgen-treatedfemales. The increased ODC activity induced by testosterone in the tubules isassociated with a concomitant increase in both ODC mRNA ( Fig. 1 ) and protein levels( Fig. 2 ). This is in agreement with in situ hybridization experiments( 20 ). In female mice treatedfor 5 days with testosterone, microscopic observations revealed that ODC wasexclusively expressed in PCT, CPST, and OSPST cells. The distribution alongthe proximal tubule of ODC mRNA (vizualized by in situ hybridization) and ODCactivity in testosterone-treated female mice completely differed from thatobserved in untreated male mice( 20, 21 ). Indeed, in adult malekidneys, physiological testosterone levels preferentially induce ODC geneexpression in the PCT, not in the straight portions of the proximal tubule( 20 ). However, this is clearly not the case in testosterone-treated female mice, raising the question ofwhether the preferential overexpression of the ODC gene in the PST was due tosupraphysiological levels of testosterone.0 B+ C9 U* w8 v

Physiologically, the high ODC activity detected in PCT and PST totallydepends on the availability of the substrate L -ornithine. Severalpotential sources of L -ornithine can be considered: 1 ) L -ornithine is present in the blood, which is filtered in theglomerulus and reabsorbed along the PCT; 2 ) L -ornithine can be taken up via the basolateral carriers of PCT and PST cells; 3 ) L -ornithine is produced from L -arginine and L -glycine by the enzyme glycine amidino transferase (EC 2.1.4.1  ),which has been localized in the proximal tubule (unpublished data); and,finally, 4 ) intracellularly, arginase AII, highly active in thefemale mouse PST, converts L -arginine into urea and L -ornithine (Levillain O, unpublished observations). It is likely that, in vivo, L -ornithine is supplied to ODC in sufficient amountsfrom these different sources and that L -ornithine availability doesnot constitute a rate-limiting factor for Put synthesis. As ODC activity wasincreased in isolated PCT and PST from androgen-treated mice, a major increasein Put synthesis was also expected in these tubules. Although we measured thethree main polyamines in whole kidneys rather than in isolated tubules, variations in renal Put levels are likely to reflect the physiological ODCactivity of PCT and PST in vivo. As expected, we observed a drastic increasein Put levels in female kidneys on the first 2 days of hormonal treatment. Thelack of a tight correlation between ODC activity and renal Put level between days 3 and 5 could be explained as follows. Put could beexported, then stocked in the blood or excreted in urine, and therefore could be absent from kidney cells( 32 ). In addition, Put couldbe metabolized into Spd and Spm or decarboxylated into GABA( 6 ). Precise localization alongthe nephron of the different enzymes involved in polyamine metabolism wasessential to elucidate the fate of Put in the proximal tubule.( \3 M# ]- |2 Q  q# k) q
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In the adult male mouse, physiological testosterone levels specifically regulate ODC gene expression in the proximal tubule. Until now, however,whether testosterone also regulates N 1 -SSAT gene expression has remained unknown. Our data clearly show that the level of N 1 -SSAT mRNA did not differ in the whole kidney ofuntreated mice. Because the female mouse does not synthesize testosterone, itseems unlikely that this androgen hormone regulates N 1 -SSAT gene expression. An alternative possibility mightbe that testosterone regulates N 1 -SSAT gene expression inmales, whereas, in the absence of testosterone, another hormone or unknownfactor controls N 1 -SSAT gene expression in females. Arecent study supports this idea. N 1 -SSAT gene expressiondoes seem to be regulated by testosterone in male mice, as N 1 -SSAT mRNA levels decreased dramatically in castrated male mice and were restored to the level of uncastrated male mice aftertestosterone injection ( 3 ). Inaddition, we show here for the first time that injection of pharmacologicaldoses of testosterone to female mice enhances the levels of N 1 -SSAT mRNAs in the kidney. Taken together, these resultsstrongly suggest that N 1 -SSAT gene expression is under thecontrol of testosterone, at least in part.+ z. w1 C( C* @6 N+ T, @. V) `
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The main physiological role of AZ1 is thought to be the control of ODCprotein levels by feedback regulation( 11 ). However, it issurprising to find similar amounts of both AZ1 mRNAs and protein in untreatedmale and female mouse kidneys. Indeed, a high level of ODC activity is presentin male kidney ( 3, 12, 33 ) and nephron( 21 ), whereas no ODC activityis detectable in the nephron of untreated female mice( 20 ). Further experiments areneeded to understand why AZ1 levels are the same in male and female mousekidney. The second surprising result is the progressive increase in AZ1 mRNAand protein levels during the course of hormonal treatment. This resultsuggests that AZ1 gene expression is regulated either directly by testosterone or indirectly by polyamines resulting from testosterone-stimulated ODCactivity. However, because AZ1 synthesis is known to be inhibited bycycloheximide and not by actinomycin D( 23 ), regulation of AZ1 geneexpression by testosterone is likely to take place at the posttranscriptionallevel.; K. G/ T4 E# o: ^) K
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Our study reveals that the N 1 -SSAT gene was equallyexpressed in male and female mouse kidney, whereas the main gender difference lay with ODC gene expression. The physiological consequence of a lack of ODCexpression in the female nephron is a low content of Put in the kidney. If Putis essential for physiological events and/or for Spd and Spm synthesis inrenal cells, it has to be synthesized and carried to renal cells from other tissues. Whether the female mouse nephron metabolizes Put into higherpolyamines remains unknown, but if not, Spd and Spm have to be transportedinto renal cells. One of these two scenarios must clearly occur, becausesimilar high levels of Spd and Spm were found in male and female kidneys. Thepresence of N 1 -SSAT and PAO in male and female kidneys( 14 ) indicates that catabolismof Spd and Spm takes place and leads to Put production. However, it seems thatthe interconversion pathway is not sufficient to compensate for the lack ofODC activity and to supply enough Put to the female mouse kidney.( s$ E* [; T5 ]% i; l& `
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In conclusion, our study has unraveled new mechanisms by which testosteroneregulates the renal polyamine pathway. Testosterone induces a differentialoverexpression of ODC in PCT and CPST and probably acts by differentmechanisms to regulate ODC gene expression in the two tubules. Testosteronelevels (physiological vs. pharmacological) may be a decisive factor in theinduction of ODC gene expression in the early and late portion of the proximaltubule. How testosterone enhances AZ1 and N 1 -SSAT mRNAlevels remains to be elucidated.
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DISCLOSURES4 w* _# y' ~6 S5 B
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This work was supported in part by the "Association pour la Recherchesur le Cancer" (contract 6083) and presented at the Gordon ResearchConference on Polyamines (August 22-29, 1999, Oxford, UK) and at the 6thEuropean Congress of Endocrinology (April 26-30, 2003, Lyon, France).

ACKNOWLEDGMENTS

The authors are indebted to Dr. Philipp Coffino (Univ. of California, SanFrancisco, CA); Dr. Leila Kauppinen (Univ. of Helsinki, Helsinki, Finland);Drs. Catherine Coleman and Anthony E. Pegg (Pennsylvania State Univ., Hershey,PA); and Senya Matsufuji (Jikei Univ. School of Medicine, Tokyo, Japan), whokindly provided, respectively, the pOD 48 plasmid containing mouse ODC cDNA,the pcDNA3.1( )-WTAZ plasmid containing mouse AZ1 cDNA, the pB-SAT9.3 plasmidcontaining human N 1 -SSAT cDNA, and the anti-AZ1 antibody.O. Levillain is indebted to Dr. J. J. Madjar, who kindly gave access to hislaboratory; Dr. John L. A. Mitchell for stimulating discussions; Dr.Emmanuelle Caron and Valérie Stiegler for improving the manuscript; Prof. Jean François Nicolas and Marie Thérèse Ducluzeau,who kindly gave access to materials for RNA extraction; Jocelyne Vial andNathalie Zemma for assistance; and Bernard Marchand for the contribution inthe animal facility.
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